Ni-NTA Fast Start Kit

E. coli溶解物からの組み換えHisタグ付きタンパク質の精製および検出用

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Ni-NTA Fast Start Kit (6)

Cat. No. / ID: 30600

6つの6xHisタグ付きタンパク質調製物の精製および検出用：6 x Fast Start Columns、Penta·His Antibody、バッファー、および試薬

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￥44,500

Ni-NTA Fast Start Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

タンパク質科学については初心者の研究者に最適
効率的な精製に必要なすべてが1つのキットに
わかりやすいプロトコールと簡単な処理
わずか90分でカラムあたり最大25 mgのタンパク質

Product Details

Ni-NTA Fast Start Kitには、充填済みNi-NTAカラムなど、澄明な E.coli溶解物からHisタグ付きタンパク質を迅速かつ効率的に精製するために必要なすべてが含まれています。キットに含まれるバッファーにより、タンパク質を天然または変性条件下で精製できます。キットには、発現したHisタグ付きタンパク質を検出するための抗His抗体も含まれています。

Performance

Ni-NTA Fast Start Kitには、澄明なE.coli溶解物からHisタグ付きタンパク質を迅速かつ効率的に精製するために必要なすべてが含まれています。キットに含まれるバッファーにより、タンパク質を天然または変性条件下で精製できます（図）。シンプルでわかりやすいプロトコールとすぐに使える試薬により、Ni-NTA Fast Start Kitは、タンパク質発現の分野に研究を広げたいと考えているもののタンパク質精製手順に詳しくない科学者にとって理想的な出発点となります。

See figures

天然または変性条件下での高純度タンパク質

Principle

Ni-NTA Fast Start Kitは、6個以上のヒスチジン残基（連続または交互）のアフィニティタグ（Hisタグ）を含むタンパク質に対する特許取得済みのNi-NTA（ニッケル-ニトリロ三酢酸）樹脂の優れた選択性に基づいています。この技術により、天然または変性条件下で、ほぼすべてのHisタグ付きタンパク質をワンステップ精製できます。ニッケルイオンに対して4つのキレート部位を持つNTAは、金属イオンとの相互作用に利用できる部位が3つしかない金属キレート精製システムよりもしっかりとニッケルに結合します。余剰なキレート部位がニッケルイオンの浸出を防ぐため、他の金属キレート精製システムを使用した場合よりも高純度のタンパク質調製物が得られます。Hisタグは、キットに付属のPenta·His Antibodyによって認識されるエピトープも生成します。

Procedure

細胞を付属の溶解緩衝液で溶解し、遠心分離して澄明な溶解物を入手します。この溶解物をFast Start Columnにかけます。カラムでは、Hisタグ付きタンパク質が高い親和性で結合し、タグなしタンパク質は通過します。洗浄後、精製タンパク質をイミダゾールを含むバッファー（天然条件）または低pHバッファー（変性条件）を用いて溶出します（図を参照）。精製手順とタンパク質の収量は、SDS-PAGE、ウェスタンブロッティング、付属のPenta-His Antibodyを用いる免疫検出でたどることができます。

See figures

高純度Hisタグ付きタンパク質。

Applications

Ni-NTA Fast Start Kitは、以下を含むあらゆるアプリケーションに適した6xHisタグ付きタンパク質を高い信頼性でワンステップ精製します。

機能研究
抗体を産生するための免疫付与
タンパク質-タンパク質およびタンパク質-DNA相互作用を含むアッセイ
構造研究

Supporting data and figures

高純度Hisタグ付きタンパク質。

Specifications

Features	Specifications
Applications	プロテオミクス
Support/matrix	Ni-NTAマトリックス
Processing	手動
Binding capacity	5～20 mg/ml
特殊機能	Ni-NTA技術
Gravity flow or spin column	自然落下
Start material	細胞溶解物
Tag	6xHisタグ
Yield	カラムあたり<10 mgタンパク質

Resources

Download Safety Data Sheets for QIAGEN product components.

FAQ

We recommend to use the EasyXpress Linear Template Kit Plus to generate PCR products optimized for use in protein expression with the EasyXpress Insect Kit II.

This kit uses specially designed primers to amplify coding DNA sequence and supplement it with regulatory elements required for optimal transcription and translation in cell-free expression systems. In addition, specially designed 5' untranslated regions (UTRs) on the sense adapter primer sequences reduce the formation of secondary structure in the translation initiation region, one of the commonest causes of low expression rates. A His-or Strep-tag II can be added to either terminus, greatly simplifying protein purification and detection after expression.

FAQ ID -1221

The buffers of the Ni-NTA Fast Start Kit are based on recipes for the respective buffers for purification of 6xHis-tagged proteins under native or denaturing conditions listed in the QIAexpressionist handbook. Specific components have been added for optimized performance. The exact composition of the buffers in the Ni-NTA Fast Start Kit is confidential. However, the buffers listed in the Appendix Section of the QIAexpressionist are compatible with the Ni-NTA Fast Start Kit, and can also be used.

FAQ ID -791

The Fast Start Columns in the QIAexpress Ni-NTA Fast Start Kit are prepacked with Ni-NTA Superflow resin.

FAQ ID -836

Imidazole does not interfere with most downstream applications and therefore does not need to be removed. If it is necessary to remove the imidazole (e.g., for some sensitive enzyme assays), it can be easily achieved by dialysis, precipitation (e.g., ammonium sulfate), or ultrafiltration.

FAQ ID -91

FEATURES	BENEFITS
The interaction of the 6xHis tag with Ni-NTA matrices is conformation independent	One-step purification can be carried out under native or denaturing conditions
Mild elution conditions can be used	Binding, washing, and elution are highly reproducible, and have no effect on protein structure. Pure protein products are ready for direct use in downstream applications
The 6xHis tag is much smaller than other commonly used tags	6xHis tags can be used in any expression system. The Tag does not interfere with the structure and function of the recombinant protein
The 6xHis tag is uncharged at physiological pH	The 6xHis tag does not interfere with secretion
The 6xHis tag is poorly immunogenic	The recombinant protein can be used without prior removal of the tag as an antigen to generate antibodies against the protein of interest
Using Factor Xa Protease, 6xHis tag can be easily and efficiently removed	The detagged protein can be used for crystallographical or NMR studies where removal of the 6xHis tag may be preferred
Some QIAexpress vectors feature a 6xHis-dihydrofolate reductase tag (6xHis-DHFR tag)	Small peptides fused to the 6xHis DHFR tag are stabilized while being expressed. The 6xHis-DHFR tag is not highly immunogenic in mouse and rat, so that peptides fused to the tag can be used directly for immunizations or epitope mapping

FAQ ID -193

The binding capacity of Ni-NTA Agarose is the same regardless of the format used. However, the batch procedure (mixing the Ni-NTA resin with lysate or protein sample prior to loading it onto a column, as opposed to loading the sample onto a column pre-packed with Ni-NTA resin) can provide more efficient binding for dilute proteins, since binding can be carried out for an extended period (approximately 1 hour), and resin amounts can be scaled for variable amounts of lysate/protein sample.

FAQ ID -147

The composition of the elution buffer in the Ni-NTA Fast Start Kit is 50 mM Na-phosphate, 300 mM NaCl and 250 mM imidazole at pH 8.0.

FAQ ID - 3353

Yes, Ni-NTA Agarose and Superflow will bind a 6xHis-tag whether it is located internally or at the C- or N-teminal end of the protein. Note that the His-tag must be exposed for binding at the surface of the protein to allow for efficient purification under native conditions.

FAQ ID -496

Compatibility of reagents with Ni-NTA matrices

Reagent	Effect	Comments
Buffer reagents
Tris, HEPES, MOPS	Buffers with secondary or tertiary amines will reduce nickel ions	Up to 100 mM has been used successfully in some cases Sodium phosphate or phosphate-citrate buffer is recommended
Chelating reagents
EDTA, EGTA	Strip nickel ions from resin	Up to 1 mM has been used successfully in some cases, but care must be taken
Sulfhydril reagents
beta-mercaptoethanol	Prevents disulfide cross-linkages	Up to 20 mM
DTT, DTE	Low concentrations will reduce nickel ions	A maximum of 1 mM may be reduce nickel ions used, but beta-mercaptoethanol is recommended
Detergents
Nonionic detergents (Triton, Tween, NP-40, etc.)	Removes background proteins and nucleic acids	Up to 2% can be used
Cationic detergents		Up to 1% can be used
CHAPS		Up to 1% can be used
Anionic detergents (SDS, sarkosyl)		Not recommended, but up to 0.3% has been used success-fully in some cases
Denaturants	Solubilize proteins
GuHCl		Up to 6 M
Urea		Up to 8 M
Amino acids
Glycine		Not recommended
Glutamine		Not recommended
Arginine		Not recommended
Histidine	Binds to Ni-NTA and competes with histidine residues in the 6xHis tag	Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged protein from the Ni-NTA matrix
Other additives
NaCl	Prevents ionic interactions	Up to 2 M can be used, at least 300 mM should be used
MgCl₂		Up to 4 M
CaCl₂		Up to 5 mM
Glycerol	Prevents hydrophobic interaction between proteins	Up to 50%
Ethanol	Prevents hydrophobic interactions between proteins	Up to 20%
Imidazole	Binds to Ni-NTA and competes with histidine residues in the 6xHis tag	Can be used at low concentrations (20 mM) to inhibit non-specific binding and, at higher concentrations (>100 mM), to elute the 6xHis-tagged
Sodium bicarbonate		Not recommended
Hemoglobin Ammonium Citrate		Not recommended Not recommended Up to 60mM has been used successfully

FAQ ID -49

To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.

Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."

Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.

FAQ ID -788